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Promoter Methylation of the SALL2 Tumor Suppressor Gene In&Nbsp MOLECULAR ONCOLOGY 7 (2013) 419e427 available at www.sciencedirect.com www.elsevier.com/locate/molonc Promoter methylation of the SALL2 tumor suppressor gene in ovarian cancers Chang K. Sunga, Dawei Lia, Erik Andrewsa, Ronny Drapkinb,c, Thomas Benjamina,* aDepartment of Microbiology and Immunobiology, Harvard Medical School, NRB-939, 77 Avenue Louis Pasteur, Boston, MA, USA bDana Farber Cancer Institute, Department of Medical Oncology, Center for Molecular Oncologic Pathology, Boston, MA, USA cBrigham and Women’s Hospital, Department of Pathology, Boston, MA, USA ARTICLE INFO ABSTRACT Article history: The SALL2 gene product and transcription factor p150 were first identified in a search for tu- Received 16 October 2012 mor suppressors targeted for inactivation by the oncogenic mouse polyoma virus. SALL2 has Received in revised form also been identified as a cellular quiescence factor, essential for cells to enter and remain in 28 November 2012 a state of growth arrest under conditions of serum deprivation. p150 is a transcriptional ac- Accepted 29 November 2012 tivator of p21Cip1/Waf1 and BAX, sharing important growth arrest and proapoptotic properties Available online 12 December 2012 with p53. It also acts as a repressor of c-myc. Restoration of SALL2 expression in cells derived from a human ovarian carcinoma (OVCA) suppresses growth of the cells in immunodeficient Keywords: mice. Here we examine the pattern of p150 expression in the normal human ovary, in OVCA- SALL2 derived cell lines and in primary ovarian carcinomas. Immunohistochemical staining Ovarian cancer showed that p150 is highly expressed in surface epithelial cells of the normal human ovary. Tumor suppressor Expression is exclusively from the P2 promoter governing the E1A splice variant of p150. The Promoter methylation P2 promoter is CpG-rich and susceptible to methylation silencing. p150 expression was re- Polyoma T antigen stored in OVCA cell lines following growth in the presence of 5-azacytidine. In a survey of 210 cases of OVCA, roughly 90% across major and minor histological types failed to show ex- pression of the protein. Immunological and biochemical approaches were used to show hy- permethylation of the SALL2 P2 promoter in OVCA-derived cell lines and in a majority of primary tumors. These results bring together molecular biological and clinical evidence in support of a role of SALL2 as a suppressor of ovarian cancers. ª 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 1. Introduction histological type (Karst and Drapkin, 2010; Kurman and Shih Ie, 2010; Vaughan et al., 2011). They may arise from the ovarian Epithelial ovarian cancers are a major cause of cancer-related surface epithelium, from the epithelium of the distal fallopian death among women. These cancers comprise a heteroge- tube, the secondary Mullerian system, or from the mesothelial neous group with respect to tissue and cell type of origin and lining of the peritoneal cavity (Bowen et al., 2009; Crum et al., Abbreviations: OVCA, ovarian carcinoma; FFPE, formalin-fixed paraffin-embedded; HOSE cells, human ovarian surface epithelial cells; Me-DIP, methylation sensitive DNA immunoprecipitation. * Corresponding author. E-mail address: [email protected] (T. Benjamin). 1574-7891/$ e see front matter ª 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molonc.2012.11.005 420 MOLECULAR ONCOLOGY 7 (2013) 419e427 2012; Dubeau, 2008). A variety of genetic and epigenetic in the normal mouse ovary, the specific cell types within the changes have been found in these cancers, some correlated ovary which express the protein and which of the two known with particular stage, grade or histological type (Asadollahi isoforms (splice variants) (Ma et al., 2001) are expressed remain et al., 2010; Cannistra, 2004; Chen et al., 2003; Cho and Shih unknown. The present investigation was undertaken to deter- Ie, 2009; Etemadmoghadam et al., 2009; Jazaeri, 2009; Lengyel, mine the pattern of SALL2 expression in the normal human 2010; Mok et al., 2009; Pearce et al., 2009; Tycko, 2000). Muta- ovary and in epithelial ovarian cancers. tions in p53 are found in a majority of ovarian cancers as well as in precancerous lesions in the distal portion of the fallopian tube (Crum, 2009; Crum et al., 2007). In particular, 96% of high- 2. Material and methods grade serous carcinomas, the most frequent and highly malig- nant type, harbor p53 mutations (2011; Engler et al., 2012). 2.1. Cells First identified as a binding target of the SV40 large T (tumor) antigen (Lane and Crawford, 1979; Linzer and Levine, 1979), p53 HOSE cells were telomerase-immortalized, HPV E6- is also targeted for inactivation or destruction by the high risk transformed human ovarian surface epithelial cells (Clauss human papilloma viruses as agents of cervical cancer and by et al., 2010; Drapkin et al., 2005). OVCA-derived cells SKOV-3 certain members of the adenovirus group that are oncogenic and RMUGS were from the American Type Culture Collection. in experimental animals (Howley and Livingston, 2009). Sur- Cells were grown in DMEM with 10% fetal bovine serum. prisingly, the highly oncogenic mouse polyoma virus stands RMUGS and SKOV-3 were subjected to DNA demethylation apart from other DNA tumor viruses, including the closely re- by incubation with 5-azacytidine (2 mM) for 5 days. lated SV40, in failing to target p53 (Dey et al., 2000). A ‘tumor host range’ selection procedure was devised with the aim of uncovering other possible tumor suppressor gene(s) with 2.2. Antisera which polyoma virus must interact in order to replicate and in- duce tumors in the mouse. The p150 product of the SALL2 gene Polyclonal antisera were raised in rabbits against the N-termi- was uncovered as a binding partner of the polyoma virus large nus (amino acids 1e550) and the C-terminus (amino acids T antigen using this procedure (Li et al., 2001). Viral DNA repli- 717e1005) of human p150 purified as GST fusions. Antisera cation is inhibited by p150 and binding by the large T protein were purified by flow through over a GST column and binding overcomes this inhibition. A virus mutant unable to bind to Staph A agarose beads. Polyclonal antisera were raised in p150 is unable to replicate and fails to induce a broad spectrum chickens against peptides representing alternative exons of tumors in the mouse (Li et al., 2001). E1A (AHESERSSRLGVPC) and E1 (QLISDCEGPSASEN). Members of the SALL (Spalt-like) gene family are evolution- arily conserved orthologues of the homeotic gene Spalt in Dro- 2.3. Tissue samples and immunohistochemistry sophila (Jurgens, 1988). They encode multi-zinc finger transcription factors with roles in embryonic development After institutional review board approval, sections of in vertebrate as well as invertebrate species (de Celis and formalin-fixed, paraffin-embedded (FFPE) human ovarian can- Barrio, 2009; Sweetman and Munsterberg, 2006). Mutations cers were obtained from the Department of Pathology at the in SALL1 (Kohlhase et al., 1999) and SALL4 (Al-Baradie et al., Brigham and Women’s Hospital (Boston, MA). All major histo- 2002; Kohlhase et al., 2005) give rise to developmental defects logical subtypes (serous, endometrioid, mucinous, clear cell, in man. SALL4 is important in maintaining a pluripotent state and transitional) were examined for p150 expression by im- in mouse embryonic stem cells (Yang et al., 2010; Zhang et al., munohistochemistry (IHC) as previously described (Clauss 2006; Zhou et al., 2007). The SALL2 transcription factor binds to et al., 2010; Drapkin et al., 2005). A high-density tissue micro- the neurotrophin receptor and plays a role in neuronal devel- array (TMA) composed of high-grade serous ovarian carcino- opment (Pincheira et al., 2009). SALL2 is essential for inducing mas (Clauss et al., 2010; Liu et al., 2009) was also utilized. and maintaining a quiescent state in human fibroblasts under Antibody to the C-terminus of p150 was used at a dilution of conditions of serum deprivation (Liu et al., 2007). It is the only 1:3000 with heat-induced epitope retrieval. Negative controls member of the SALL family implicated as a tumor suppressor included protein-A purified preimmune serum, secondary an- (Li et al., 2001; Ma et al., 2001). tibody alone, and anti-p150 antibody preincubated with Though unrelated to p53 in amino acid sequence and differ- recombinant p150 protein. Control sections were prepared ing in its DNA binding specificity (Gu et al., 2011), p150 overlaps from normal ovaries obtained from women with benign gyne- functionally with p53 in inducing expression of p21Cip1/Waf1 cologic diseases under protocols approved by the institutional and BAX (Li et al., 2004). Unlike p53, p150 is not a DNA damage review boards of the Brigham and Women’s Hospital. None of response protein but is stably and highly expressed in certain the negative controls generated a positive signal in IHC. terminally differentiated cells. In a survey of normal mouse tissues, the highest level of expression was found in the ovary (Li et al., 2001). Restoration of SALL2 expression in an OVCA- 2.4. Western blots derived cell line deficient in p150 expression resulted in partial suppression of tumor growth in SCID mice. Suppression of Immunoblotting was carried out on cell extracts using an Od- growth was accompanied by a decreased mitotic index and yssey infrared imaging system (LI-COR Biosciences, Lincoln, an increased apoptotic index along with induction of p21Cip1/ NE). Intensity values were determined with LI-COR Odyssey Waf1 and BAX (Li et al., 2004). While p150 is highly expressed software (Li-COR Biosciences). MOLECULAR ONCOLOGY 7 (2013) 419e427 421 2.5. Assays for methylated DNA 3. Results Me-DIP (methylated DNA immunoprecipitation) assay was carried out on purified DNAs (1 mg) with methylCpG-specific 3.1. The E1A splice variant of p150 is expressed in monoclonal antibody conjugated magnetic beads from Active established human ovarian surface epithelial cells Motif (Carlsbad, CA) following manufacturer’s suggestions.
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